Oncogenic transformations are closely correlated with the change of glycosylation patterns of cell surfaces, and the aberrant carbohydrates expressed on tumors, which are called tumor-associated carbohydrate antigens (TACAs), are important targets for the development of cancer vaccines that can be used for cancer diagnosis and therapy. However, the problem of immunotolerance to TACAs has severely hindered further progress in the area. To solve this problem and to develop new, effective cancer vaccines, this project will exploit a new strategy that is based on modified TACAs and metabolic engineering of cancers. First, a TACA on cancer cells will be metabolically modified by treating them with an artificial derivative of a monosaccharide that can be taken as a precursor by cancer cells to biosynthesize a neoantigen - an artificially modified analog of the TACA. The metabolic engineering of cancer takes the advantage of the remarkable flexibility of carbohydrate biosynthetic machineries. Then, specific monoclonal antibodies (mAbs) will be used to selectively target tumor cells labeled by the neoantigen. The mAbs in return can be prepared with a synthetic vaccine made of the neoantigen. As vaccines made of artificial carbohydrates are potentially more immunogenic than those made of the natural TACAs and the metabolic engineering can specifically mark cancer cells, effective vaccines may be easily composed from modified TACAs and the new strategy will potentially solve the problem of immunotolerance to TACAs. The metabolic modifying targets of this project are sialyl TACAs. Melanoma is employed as the tumor model, and GD3 and GM3 on its cells are the specific targets. The N-modified analogs of mannosamine and sialic acid will be used as the precursors, and the neoantigens expressed on melanoma will then be the N-modified derivatives of GD3 and GM3. The aims of this project are: 1)to find the effective precursors for metabolic engineering of melanoma via studying the bioavailability of various precursors to the enzymes involved in the biosynthesis of sialyl TACAs and to melanoma cells; 2) to find the effective vaccines that can provoke specific immune responses to the neoantigens via studying the conjugates of various N-modified GD3 and GM3; 3) to illustrate the new strategy through specific immunotargeting of metabolically engineered melanoma. This research will eventually establish a proper combination of the precursor and vaccine. As the overexpression of sialic acid is found in various tumors and sialic acid is a shared feature of many TACAs, the principles established herein may be of wide applicability.